The mechanism of oxygen activation by tetrahydrobiopterin and dihydroflavin mixed function hydroxylases is unknown. Our recent findings, that appropriately substituted pyrimidines can serve as cofactors for the tetrahydrobiopterin dependent hydroxylases, has provided us with a new approach to this problem. Pteridine and pyrimidine analogs have been designed to determine (a) whether an oxygen cofactor adduct is formed, (b) the structure of the activated oxygen complex, (c) the contribution of cofactor substituents to the formation and effectiveness of the complex, (d) the nature of the forces binding cofactor to the catalytic site and their effect on function, and (e) the tautomeric form of the initial dihydropterin product. The structural features that distinguish tetrahydrobiopterin from dihydroflavin will be related to the differences in their properties and functions. In comparison to dihydroflavin dependent enzymes, phenylalanine hydroxylase has the advantage that cofactor freely dissociates from enzyme, cofactor structures are easily modifiable, overall reaction is slow, and cofactor is relatively stable to autooxidation. A new assay which we have developed, for determination of tyrosine formed in a phenylalanine hydroxylase reaction, allows detection of cofactor activity of compounds more than four orders of magnitude slower than tetrahydrobiopterin. It is hoped that phenylalanine hydroxylase will provide a model of oxygen activation, not only for the three aromatic amino acid hydroxylases, but also for the external dihydroflavin monooxygenases. Elucidation of the catalytic function of tetrahydrobiopterin will provide a basis for the understanding of its role in the regulation of tyrosine and tryptophan hydroxylases, the rate limiting enzymes in the synthesis of serotonin and the catecholamine neurotransmitters and hormones.